The majority of human genes have poorly characterized function. Naturally occurring loss of function variants (LoFs) provide insight into the phenotypic impact of gene inactivation. Homozygous LoFs are responsible for many rare diseases, but are also present in healthy adult individuals. The examination of such variants in health and disease can provide insight into human gene function. To explore the impact of homozygous LoFs on human phenotypes we have exome sequenced 2,625 individuals, ascertained as fit adults, with a range of parental relatedness. We developed an algorithm to detect autozygous stretches and observed 165 (6%) samples with >12.5% autozygosity (expected of double first cousin relatedness) and an additional 689 (26%) individuals with autozygosity around 6.25% (first cousins). The remainder had a range of more distant relatedness around 3-4%. From estimates across all individuals we find that essentially every site in the coding region of the genome can be effectively homozygozed in healthy individuals. We observed 657 rare (<1%) LoF homozygotes in 639 genes, 96.4% within long autozygous sections. Population analysis revealed a total of 17,520 LoFs, on average 148 per individual, including 40 homozygous; 0.5 of them rare. Rare knockout genes include single gene drug targets in current preclinical to phase II development and 43 genes where knockout of the mouse homolog is lethal. Additionally, we observe knockouts in genes present in high penetrance Mendelian diseases having no apparent effect in these fit adult samples, thus providing potential information about their penetrance. By measuring the reduction in rare LoF mutation density in autozygous stretches compared to that in heterozygous regions, we have been able to estimate the fraction of genes for which loss of function is incompatible with healthy life. We describe large-scale validation experiments for all observed knockouts at the DNA level, and planned analyses at the RNA, protein and functional levels. We are now expanding beyond this pilot to the sequencing of 15,000 individuals with self-stated parental relatedness with consent to recontact for deeper phenotyping, allowing us to investigate in vivo the phenotypic consequences of knockouts in a large number of genes. Based on our pilot data we expect to find knockouts in a third of human genes, including multiple knockouts in many cases, providing phenotypic annotation for many currently uncharacterized human genes.